DESCRIPTION (from applicant's abstract): The overall goal of the present proposal is to understand the cellular mechanisms by which beta-endorphin (betaEND) neurons control hypothalamic neurosecrelory cell activity, and Ultimately the neurosecretion of hypothalamic peptides and amines in the female. We will use the guinea pig as a model because its ovulalory cycle mimics the Human, and physiologicaal levels of E2 rapidly uncouple mu-opioid and GABAbeta receptors from K+ channels (lKir) in betaEND neurons via a protein kinase pathway. In Experiments 1, we will test the hypothesis that E2 activates a protein kinase C (PKC) pathway to uncouple mu-opioid and GABAbeta receptors from lKir in arcuate (ARC) neurons. We will measure: (a) the changes in the potency of mu-opioid and GABAbeta agonists in ovaricetomized females treated acutely with E2 using PKC activators and inhibitors; (b) elucidate the pathway by which longer-term (24 h) E2 uncouples mu-opioid and GABAbeta receptors; and (c) changes in expression of regulators of G-protein signaling (RGS) proteins using in situ hybridization and ribonuclease protection assay following E2 treatment. In Experiments 2, we will determine to which effector systems mu-opioid and orphanin FQ receptors are coupled in supraoptic (SON) oxytocin and vasopressin neurons and the effects of long term E2 treatment. We will: (a) further characterize the inhibition of lh by mu-opioid agonists and ascertain the specific Ca2+ conductance (s) inhibited by K-opioid agonists; and the specific K+ conductance(s) activated by OFQ; (b) ascertain the effects of E 2 on the mu-opioid, k-opioid and OFQ responses in SON neurons; (c) characterize the opioid synaptic synaptic input to SON neurons in E2-treated animals; and receptor autoradiography. In Experiments 3, we will use a strain of gene-targeted betaEND deficient (beta END Knockout, KO) mice to ascertain if there is a decrease in mu-opioid receptor coupling induced by the absence of an endogenous opioid. We will: (a) investigate the changes in mu-opioid receptor coupling to lkir in arcuate neurons; (b) measure changes in the presynaptic actions of mu-opioids to inhibit excitatory input to ARC neurons, (C) measure the effects of E2. to after the coupling of mu-opioid receptors to lkir; and finally (d) to measure the potency and efficacy of a GABAbeta agonist to aviate lkir in KO mice. These results will elucidate the cellular mechanisms by which E2 alters the coupling of the mu-opioid receptor to its effector system, which ultimately determinesmu-opioid tone in the female CNS. Also the results will help us understand the role betaEND neurons in the control of homeostasis.